Posttranslational modification of eukaryotic proteins, particularly therapeutic proteins such as immunoglobulins, is often necessary for proper protein folding and function. Because standard prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in production of these therapeutic proteins. Even where eukaryotic proteins do not have posttranslational modifications, prokaryotic expression systems often lack necessary chaperone proteins required for proper folding. Yeast and fungi are attractive options for expressing proteins as they can be easily grown at a large scale in simple media, which allows low production costs, and yeast and fungi have posttranslational machinery and chaperones that perform similar functions as found in mammalian cells. Moreover, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells as well as more complex eukaryotic cells such as mammalian or insect cells (De Pourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31).
However, posttranslational modifications occurring in yeast and fungi may still be a concern for the production of recombinant therapeutic protein. In particular, insufficient N-glycosylation is one of the biggest hurdles to overcome in the production of biopharmaceuticals for human applications in fungi.
N-glycosylation, which refers to the attachment of sugar molecule to a nitrogen atom of an asparagine side chain, has been shown to modulate the pharmacokinetics and pharmacodynamics of therapeutic proteins.
Hintz et al (1995, Can. J. Bot. (Suppl 1): S876-S884) report genetic engineering of filamentous fungal cells such as Aspergillus nidulans and possible strategies for remodeling N-glycans in such host cells. Contreras et al disclose strategies for producing glycoproteins with mammalian-like N-glycans in yeast Pichia, comprising the overexpression of α1,2 mannosidae and α-glucosidase II enzyme (US2010/0267084). De pourcq et al further report the production of Man3GlcNAc2 N-glycan core in yeast Yarrowia lipolytica, by disrupting Alg3 gene, and overexpressing A. niger α-glucosidase II and α1,2 mannosidase. US2009/0069232 and WO2011061629 further discloses genetically engineered cells of Yarrowia for producing altered N-glycosylation form.
In contrast, U.S. Pat. No. 7,491,510 reports the use of glucosidase II mutation in Trichoderma reesei strain to increase protein secretion, possibly in combination with α1,2 mannosidase and/or glucosaminyl-transferase gene. WO2012/069593 discloses improved methods for producing complex N-glycans and glycoproteins with mammalian-like N-glycans, using in particular novel recombinant GnTI and GnTII enzymes. WO2013/102674 further discloses filamentous fungal cells with reduced protease activity and their use in production of heterologous proteins. WO2013/174927 further discloses strategies to express fucosylation pathway in filamentous fungal cells and produce fucosylated glycoproteins.
Reports on filamentous fungal cell expression systems expressing human-like N-glycans are lacking. In particular, a need remains in the art for improved filamentous fungal cells, such as Trichoderma filamentous fungal cells, that can stably produce heterologous proteins with predominant mammalian-like N-glycans, such as predominant G0, G1 or G2 glycoform, and at high levels of expression.